It is well known that ovarian steroids markedly influence the proliferative behavior of the mammary gland at different stages of gland development. One or more of these steroids are responsible for the prepubertal formation of the mammary gland, the proliferation observed during the normal menstrual cycle, and the lobuloalveolar growth observed during pregnancy. Both epidemiological studies of human females and laboratory studies with rodents support that ovarian estrogens and progestins are important for the formation of mammary neoplasias. The focus of our experiments has been to understand the biochemical basis for the proliferative action of 17beta-estradiol (E2) or progesterone. We have previously demonstrated that transforming growth factor-alpha (TGF-alpha), a member of the EGF family of ligands, occurs in the proliferating epithelial cap cells during murine mammary ductal morphogenesis and also that local implants of TGF-alpha or EGF can mimic the effects of 17beta-estradiol. Ovariectomized adult female mice treated with E2 for several days exhibit end buds that resemble those observed during morphogenesis. The mammary glands of E2-treated mice also revealed increased levels of TGF-alpha transcripts as determined by reverse transcription-polymerase chain reaction (RT-PCR) when compared to controls. We are currently screening a mouse genomic library in order to determine whether estrogen response elements (EREs) exist in the 5'-flanking region of the mTGF-alpha gene. Inspection of the known human and rat TGF-alpha gene sequences suggests that two imperfect EREs exist between -191 and -237 bp from the initiation site. These potential EREs are separated by an intervening sequence of 20 bp. Extracts of nuclei obtained from uteri of E2-treated mice or yeast expressing the human estrogen receptor yields a band in gel retardation assays with a cognate 32P-oligonucleotide (53 bp) containing the two potential EREs and intervening sequence of the rat TGF-alpha gene. These data, so far, support that the activated estrogen receptor may be a positive transcriptional factor for the TGF-alpha gene. The capacity of the estrogen receptor to act as a cis-regulatory factor for an EGF- like ligand may be a mechanism used by other sex steroids to stimulate cell proliferation in target organs.